JXB Advance Access originally published online on January 30, 2004
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Journal of Experimental Botany, Vol. 55, No. 397, pp. 783-785, March 1, 2004
© 2004 Oxford University Press
GENE NOTE |
ADL2a, like ADL2b, is involved in the control of higher plant mitochondrial morphology*
Received 18 November 2003; Accepted 24 November 2003
Sir Harold Mitchell Building, School of Biology, University of St Andrews, St Andrews, Fife KY16 9TH, UK
*See note added in proof on page 3. 
To whom correspondence should be addressed. E-mail: david.logan{at}st-and.ac.uk
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Abstract |
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 Top Abstract Supplementary dataNote added in proofReferences |
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A mitochondrial–GFP construct was used to tag mitochondria fluorescently in a T-DNA knockout line for the Arabidopsis dynamin ADL2a. Visualization of mitochondria in vivo demonstrated that disruption of ADL2a affected mitochondrial morphology. Mitochondria in the mutant had a complex morphology; occasionally large spherical organelles could be seen, but, more frequently, the mitochondria adopted a tubular morphology with many constrictions along their length. Mitochondria in the mutant also frequently possessed long protuberances that were named matrixules, extending to many micrometres in length.
Key words: Dynamin, mitochondrial division, morphology, organelle fission.
Mitochondrial fission is controlled by dynamin-like proteins (Shaw and Nunnari, 2002; Logan, 2003). The Arabidopsis genome contains 16 dynamin-like genes and one of these, ADL2b (At2g14120), was recently shown to be involved in the maintenance of normal mitochondrial morphology (Arimura and Tsutsumi, 2002). This paper describes research testing the hypothesis that a second dynamin-like protein, ADL2a (At4g33650), sharing 70% amino acid identity with ADL2b, also functions in the control of mitochondrial morphology.
To identify a knockout mutant of ADL2a, the Salk sequence-indexed insertion lines (Alonso et al., 2003) were searched and one line was selected with a T-DNA left-border flanking-sequence of low e-value (SALK_066958, 6e-95). The seed obtained from the NASC stock centre was germinated on kanamycin selection media and individuals were tested by PCR to confirm the presence of the T-DNA insertion (which is in the fifth intron at the 5'-end of the gene). Gene-specific PCR primers flanking the insertion in line SALK_066958 were used in combination with a primer to the T-DNA left border (primer LBb1). Under the conditions used, PCR amplification using wild-type DNA yields a fragment only with the gene-specific forward (Gf) and reverse (Gr) primers, whilst amplification of a smaller fragment using the Gr primer and T-DNA left border primer (LBb1) indicates a T-DNA insertion within the fifth intron of ADL2a. Following PCR amplification, DNA extracted from plants hemizygous for the insertion would yield a product with either set of primers whereas DNA from plants homozygous for the insertion would only yield a product with the LBb1 and Gr primers. The progeny of these plants (an unselected population containing individuals either homozygous for the insertion, hemizygous, or containing no insertion, as determined by PCR) were genetically transformed to tag the mitochondria with GFP. Briefly, the mgfp5-atpase cDNA in the vector pBINmgfp5-atpase (Logan and Leaver, 2000) was PCR amplified, ligated downstream of the 35S CaMV promoter and cloned into the plant transformation vector pMLBART (conferring resistance to glufosinate ammonium) to create pMLBARTmgfp5-atpase. Plants were transformed with pMLBARTmgfp5-atpase by Agrobacterium-mediated transformation. T1 seeds were germinated on media containing kanamycin prior to transfer to compost. Plants in compost were sprayed with 120 mg l–1 glufosinate ammonium to select for positive pMLBARTmgfp5-atpase transformants.
Individual GFP-positive T1 plants were analysed by PCR to identify individuals hemizygous for the original SALK_066958-derived T-DNA insertion and these plants were allowed to set seed. Seeds from one of these hemizygous plants were germinated without selection and the mitochondrial phenotype of 94 individuals was analysed using an epifluorescent microscope. Of the 94 GFP-positive plants, 24 were identified that had a mutant mitochondrial phenotype with a segregation ratio of 2.9:1 (wild type:mutant). Statistical analysis of this result, using the chi-square (
2) test for goodness of fit, demonstrated that the observed results are in very good agreement (P >0.9) with the predicted phenotypic ratio of 3:1 for the segregation of a single recessive nuclear gene. The genotype with respect to the SALK_066958-derived T-DNA insertion of each of the 94 individuals was determined next by means of PCR analysis using the gene specific primers and left border primer, as described above. This analysis demonstrated that not only were all 24 individuals with abnormal mitochondrial phenotypes homozygous for the T-DNA insertion, but that plants with a wild-type mitochondrial phenotype were either hemizygous for the T-DNA insertion (50 individuals) or contained no insertion (20 individuals) (Fig. 1a; and data not shown). Statistical analysis of these results using the 2 test demonstrated that deviation from the expected 1:2:1 ratio of genotypes (wild type:hemizygote:homozygous mutant) of progeny from an inbred hemizygous individual was not statistically significant (P >0.5). This analysis confirmed that the aberrant mitochondrial phenotype was due to the presence of a homozygous T-DNA insertion within the ADL2a gene.
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Since it was expected that the T-DNA insertion would lead to the generation of a null allele through the generation of a nonsense transcript, the effect of the insertion on transcript abundance was determined next by analysis of the steady-state abundance of the mature ADL2a mRNA in two homozygous T-DNA lines and a line hemizygous for the T-DNA insertion. In order to differentiate between ADL2a and ADL2b, the probe was prepared by random priming using a PCR fragment amplified from the 3'-UTR of ADL2a. There was no detectable accumulation of the mature ADL2a mRNA in the two homozygous T-DNA lines whilst the hemizygous line had a reduced accumulation of ADL2a transcript relative to the wild type (Fig. 1b).
Mitochondrial morphology visualised in vivo in wild-type Arabidopsis is dynamic, the organelles alternating between spherical, sausage-shaped, and, occasionally, longer vermiform structures (Fig. 2a; supplementary material (movie 1) can be found at Journal of Experimental Botany online; Logan and Leaver, 2000; Logan, 2003). In lines homozygous for the T-DNA insertion in ADL2a, the mitochondrial phenotype is grossly altered: mitochondria are either large spherical organelles (rarely, Fig. 2b; supplementary material (Fig. S1) can be found at Journal of Experimental Botany online) or long tubular structures often with many constriction sites (Fig. 2c–f). Chloroplast morphology in the mutant is normal. A third mitochondrial phenotype, rarely seen in wild-type mitochondria, but frequently observed in the ADL2a knockouts, is shown in the images in Fig. 2g–j. These mitochondria are characterized by a thin protuberance extending up to many micrometers in length that, to the authors’ knowledge, have not been described before in the literature. These protuberances were termed ‘matrixules’ in keeping with the naming as stromules of similar structures, seen to extend from chloroplasts (Kohler and Hanson, 2000).
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Kang et al. (1998) have shown that heterologous over-expression of an ADL2a–GFP fusion protein in soybean or tobacco leads to the detection of GFP-fluorescence in chloroplasts. However, in Arabidopsis transiently over-expressing N-terminal ADL2a–GFP chimeras, fluorescence was detectable in the cytoplasm but there was no detectable GFP-fluorescence from the chloroplasts (Arimura and Tsutsumi, 2003). The mitochondrial morphology in ADL2a knockout mutants is similar to that in cells expressing dominant negative mutations in ADL2b (Arimura and Tsutsumi, 2002) and like ADL2b, ADL2a has been localized to the constriction sites and tips of Arabidopsis mitochondria (Arimura and Tsutsumi, 2003). Mitochondria in cells with dysfunctional ADL2b were longer and less numerous and/or formed clumps (Arimura and Tsutsumi, 2002). In the ADL2a T-DNA knockout the mitochondrial phenotype is complex. The mitochondria form long tubules with numerous constrictions along their length and there are a high proportion of mitochondria with matrixules relative to those of wild-type plants (Fig. 2). The similarity of the mitochondrial morphology in ADL2a and ADL2b mutants, as far as the formation of mitochondrial tubules is concerned, suggests that these two dynamin-like proteins perform similar, but non-redundant, functions in controlling mitochondrial division. It is proposed that the presence of mitochondria with numerous matrixules can also be attributed to the function of ADL2a as a component of the mitochondrial division machine. The hypothesis is that, prior to division, the mitochondria elongate by a process that pushes or pulls them through a constrictive collar, possibly the mitochondrial division ring (MD ring; Kuroiwa, 2000), encircling the mitochondria at one pole, which forces them to adopt an elongated morphology. The first portion of the mitochondria to be extruded in this way forms the matrixule that grows and expands as the mitochondrion passes through the collar (supplementary material (movies 1 and 2) can be found at Journal of Experimental Botany online). Upon adopting a more elongate morphology the mitochondrion is ready to undergo division (supplementary material (movie 1) can be found at Journal of Experimental Botany online). It is suggested that, in Arabidopsis mitochondria, both ADL2a and ADL2b are involved in mitochondrial division. It has been shown that following mitochondrial division in yeast, dynamin remains associated with one cut end of the mitochondrion (Legesse-Miller et al., 2003). Based on the mitochondrial morphology of the ADL2a mutant and the localization of ADL2a to the tips of mitochondria (Arimura and Tsutsumi, 2003), it is speculated that one of the roles of ADL2a is to remain associated with the mitochondrion after division, at what is now one pole of the daughter organelle, to prevent further division events that would otherwise lead to severe fragmentation of the mitochondria. The fact that fragmentation does not occur in the ADL2a knockout mutant suggests there may be reduced efficiency of the division process in the absence of ADL2a. It is postulated that in the ADL2a knockout mutant, the absence of ADL2a enables daughter mitochondria from recent division events to re-enter the division process, starting with elongation via collar-based extrusion and the initial formation of a matrixule. This hypothesis explains why, although matrixules are occasionally observed in wild-type cells, they are much more frequent in the cells of the ADL2a knockout.
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Supplementary data |
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TopAbstractSupplementary dataNote added in proofReferences |
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Two movies and Fig. S1 can be found at Journal of Experimental Botany online.
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Acknowledgements |
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This research was supported by the Biotechnology and Biological Sciences Research Council, grant numbers 49G13350 and 49G1554, and by a research studentship to IS. We thank Mr Harry Hodge for technical assistance in the laboratory, growth rooms, and glasshouse. The Arabidopsis beta-tubulin cDNAs were cloned by Dr Sarah Scrase-Field, University of Southampton. pMLBART, engineered by Bart Janssen, HFRI, Auckland, New Zealand, was a kind gift from Kathy Osteryoung, MSU, MI, USA.
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Note added in proof |
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TopAbstractSupplementary dataNote added in proofReferences |
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Hong et al. (2003) (Plant Molecular Biology 53, 261–265) have recently suggested a unified nomenclature for all 16 Arabidopsis dynamin-related proteins. We welcome this initiative under which ADL2a and ADL2b are renamed DRP3A and DRP3B, respectively.
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References |
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TopAbstractSupplementary dataNote added in proofReferences |
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Alonso JM, Stepanova AN, Leisse TJ, et al. 2003. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657.
Arimura S, Tsutsumi N. 2002. A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proceedings of the National Academy of Sciences, USA 99, 5727–5731.
Arimura S, Tsutsumi N. 2003. Mitochondrial division in Arabidopsis is performed by two dynamin-like proteins; ADL2a and ADL2b. (Abstract 1202) American Society of Plant Biologists, 2003, July 25–30, Honolulu, Hawaii; URL: http://abstracts.aspb.org/pb2003/public/P68/0189.html.
Kang SG, Jin BJ, Piao HL, Pih KT, Jang HJ, Lim JH, Hwang I. 1998. Molecular cloning of an Arabidopsis protein that is localised to plastids. Plant Molecular Biology 38, 437–447.[CrossRef][ISI][Medline]
Kohler RH, Hanson MR. 2000. Plastid tubules of higher plants are tissue-specific and developmentally regulated. Journal of Cell Science 113, 81–89.[Abstract]
Kuroiwa T. 2000. The discovery of the division apparatus of plastids and mitochondria. Journal of Electron Microscopy 49, 123–134.
Legesse-Miller A, Massol RH, Kirchhausen T. 2003. Constriction and Dnm1p recruitment are distinct processes in mitochondrial fission. Molecular Biology of the Cell 14, 1953–1963.
Logan DC. 2003. Mitochondrial dynamics. New Phytologist 160, 463–478.[CrossRef]
Logan DC, Leaver CJ. 2000. Mitochondria-targeted GFP highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. Journal of Experimental Botany 51, 865–871.
Shaw JM, Nunnari J. 2002. Mitochondrial dynamics and division in budding yeast. Trends in Cell Biology 12, 178–184.[CrossRef][ISI][Medline]
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